Abstrtlct-Bindin, of e H)-ouabain aod ouabain-induced inhibition of thc sodium pump and nf Ihe (Na" + K·}-ATPase have becn cbaracterized in cullurcd cardiac muscle aod Don musele cclls, as weil as in cardiac cell mcmbran~ll obtained from dUck embryos.

Abstract-The bindiog of (lH!ouabain has beta studied in (Na' .... K+) -ATPase enriched cardiac te il membranes, as weil as in ca rdiac musel e aod non-musele cells in culture-all oblained (rom heans of nwoatal rals. The bindiog has been correlated with ouabaio-induced inhibition of (Na' + K")-A TPase (cardiac cdl membranes) aod the inhibition of aetive (SORb" + K' )-inßux (cardiac musele sild non· musele cells in culture). Furthennore, Ihe eUeet of ouabain on the amplilude of eell-wall motion and conlraction vc:locity has been Sludied in eleelrieally driven cardiae muscJ e eells.in muscle and non-muscle eells, IWO cJasses of ouabain binding si tes have been identified. In rat heaT! muscle cells, the high affinity binding site has a dissodation constant (KD ) of 3.2 x IO -~ M and a binding capacity (8) of 0.2 pmole/mg protein (80,000 sites/eell ); the values for the low affinity bin ding site are:

The Machaniam of positive inotropic action of cardiac glycoaidea is still a matter of controvera1 (1). The disagreement aainly relatee to the queation whether increaaed force of contraction la cauaed by, and therefore neceasarily accompanied by, an inhibition of the sodium pump. At therapeutic levels of the drug . RodiuM pump has been deaeribed aa not inrlueneed, inhibited or even stimulated. depending on the apeciea invastigated, the drug applled and. the method used (1,2). The problem is further complicated by the preaenee of two different binding aites ror cardiae glycoaides. In rat heart, occupation of the high affinity site being linked to positive inotropic effect without inhibition of (Na++ K+)-ATPase aetivity or active (86Rb +)_uptake aa meaaure of sodium pump aetivity (3). In fev of the e~peri.ent6 deacribed, how ever, haa inhibition or atimulation of the aodium pump by cardiae glycoaides been correlated quantitatively with the pereentage of binding sites oeeupied b y cardiac glycoside molecu les . Methods: Huscle and non ~uscle cells from hearts of 9.12 day_old c hi ck embryos haye been prepared and separately cultured a8 described in detail for rat heart cells (4,5).Experiments have been ~arried out with museie cells after 2-~ days in c~!tu re, with non muscle cells after 1 aubcultivation (splitting rati o 1:2). At that time, muscle ce lla had forMed a synchronously beating monolayer, non musc le cells wer@ at conflue nc y. For measurement of {3H)_ouabain binding, (86Rb ++ K+)-uptake and cellular K+, cells (0.2-2.0 mg proteinjfla.sk) were incubated in Ilepes buffered (20 mM, pli 7.40)